This invention relates to novel compositions of liposomes and their use in the effective treatment of malaria. It is well known that malaria is caused by an infection of a vertebrate host with protozoan parasites, known as Plasmodia, which are transmitted by the bite of an infected anopheline mosquito. Malaria infections in man have remained a considerable cause of morbidity and mortality despite efforts to control the mosquito vector and to eradicate infections in geographic regions roughly from 60.degree.N to 40.degree.S latitude. There are four species of Plasmodia that are pathogenic to man, namely: Plasmodium falciparum; Plasmodium vivax; Plasmodium ovale; and Plasmodium malariae. Each species has a developmental cycle which is initiated by the infectious forms (sporozoites) of the organism into man by the anopheline mosquito. In the human host, the sporozoites are taken up and later invade liver cells (hepatocytes) were asexual multiplication (tissue schizogony) occurs to produce organisms called merozoites. These merozoites invade red blood cells (RBC). Within the red blood cells, the parasites again undergo asexual reproduction to produce merozoites which are liberated by rupture of the host red blood cells. This process is ordinarily associated with malaria fever. These newly formed merozoites invade new red blood cells resulting in the production of gametocytes. When a blood meal is taken by the anopheline mosquito, the asexual forms of the malaria parasite are destroyed and thereafter the gametocytes undergo maturation, fertilization, and further development in the sexual cycle (sporogony) which leads to the formation of sporozoites, the infectious forms present in the salivary gland of the mosquito. In the case of Plasmodium falciparum, there is relatively little secondary invasion of the liver by parasites. However, secondary invasion does occur with Plasmodium vivax infections and, to a lesser extent, with Plasmodium malariae and Plasmodium ovale infections. Secondary invasion of the liver may give rise to multiple recurrence of malaria attacks as a consequence of development and further tissue schizogony of the parasites.
Prior to this invention, interruption of the life cycle of malaria within the host has been achieved at several stages. Measures to control the infection by eradication of mosquitos in certain areas have had a drastic effect on potential sporogony. Sporonticides (block man/vector contact) have been used with limited success to control the number of mosquitos in highly infested areas, thereby reducing the number of infectious mosquitos which are available to attack man. The interruption of schizogony in man may be achieved in tissues (tissue schizontocides) or in the blood (blood schizontocides). Additionally, sexually-differentiated forms of the malaria parasites may be attacked with gametocytocides.
Each of the foregoing means for interrupting the plasmodial life cycle has certain shortcomings. However, in the absence of superior measures, such as a vaccine, they must be relied upon at the present time. To date, compounds which are effective against certain development stages of malaria parasites in man are known to have undersirable side effects on the host. Therefore, improvements in the treatment of malaria are being actively pursued.
Concepts on the potential applicability of liposome-targeting of drugs to malaria studies were developed based on an appreciation of the pathophysiology of malarial infection and features of its plasmodial life cycle. The parasites injected in man by the bits of the mosquito are in the form of sporozoites which travel to the liver. The organisms remain in hepatocytes for some days in the primary exoerythrocytic stage of the infection prior to emerging as exoerythrocytic schizonts and developing of a "patent" infection characterized by the appearance of parasites in erythrocytes. We have developed a novel method for "targeting" liposomes to hepatocytes.
Liposomes are defined as closed vesicles, or sacs, which contain phospholipids (examples of which are lecithin and sphingomyelin) and which may contain other lipids (examples of which are cholesterol and other sterols or steroids; charged lipids such as dicetyl phosphate and octadecylamine; glycolipids; fatty acids and other long-chain alkyl compounds; hydrophobic glycoproteins; and lipid-soluble vitamins and lipoidal surfactant-like molecules). When shaken in the presence of an excess amount of water, the lipid mixture is formed into discrete particles consisting of concentric spherical shells of lipid bilayer membranes which are referred to as multilamellar liposomes (MLL). Upon sonication, or by alternative methods of manufacture, small or large unilamellar liposomes (ULL) can be formed. In 1965, it was demonstrated that the MLL vesicle membranes were completely closed and did not allow escape of a marker compound present in the aqueous interspaces; similar properties later were found for ULL.
Numerous studies have shown that liposomes, upon injection into animals and man, are taken up rapidly by cells, and intra-cellular lysosomes, of the reticuloendothelial system, particularly those in the liver. Because of the relative impermeability of liposomes, and speedy removal of them from the circulatory system, substances (such as drugs) in the aqueous interspaces of liposomes remain concentrated and are not exposed to plasma. Moreover, there would be a strong possibility of prolonged effectiveness of a drug through slow biodegradation of the multilamellar membrane structure of the liposomes. The characteristics of liposomes thus suggest their suitability as carriers for antiparasitic agents.